A Process For Isolation Of Zeaxanthin From A Mutant Marigold Extract


Updated about 2 years ago

Abstract

The present disclosure deals with the process for isolation of zeaxanthin from a mutant marigold extract. The process provided in the disclosure is simple, safe and cost effective and can carried out from small scale to large scale. The product comprising zeaxanthin crystals prepared by the process of the present disclosure can be used for various applications including eye care supplements, nutritional supplements and pharmaceutical applications.

Information

Application ID 745/CHE/2011
Invention Field TRADITIONAL KNOWLEDGE CHEMICAL
Date of Application 2011-03-11
Publication Number 06/2014

Applicants

Name Address Country Nationality
AVT NATURAL PRODUCTS LTD. 64, RUKMANI KAKSHMIPATHY SALAI, EGMORE, CHENNAI-600 008. India India

Inventors

Name Address Country Nationality
PUSHPAKUMARI K.N. APT 16 B2, KENT HAIL GARDEN, BEHIND INTERNATIONAL STADIUM, KALOOR KOCHI-682 025. India India
PRAMOD S. 6/386, SIVA NIVAS, PURAPPILLIKAVU KARUMALLOOR P.O. N.PARUR ERNAKULAM-683 511. India India

Specification

FIELD

The present disclosure relates to a process for the isolation of free zeaxanthin. Particularly, the present disclosure relates to a process for the isolation of free zeaxanthin from mutant marigold extracts.

BACKGROUND

Health supplements have gained considerable importance over the past decade to help maintain a healthy and improved lifestyle. In recent years, carotenoids, which are C40 compounds, have emerged as an important supplement in human health. The carotenoids are divided into two classes: carotenes which are purely hydrocarbons and contain no oxygen such as a-carotene, P-carotene and lycopene and xanthophylls which contain oxygen such as lutein, astaxanthin, and zeaxanthin. The carotenoids are tetraterpenoid organic pigments which naturally occur in chloroplasts and chromoplasts of plants and have a system of extended conjugated double bonds which give these molecules color and light absorbing properties.

Lutein and zeaxanthin have been scientifically proven to reduce the risk of Age-Related Macular Degeneration of the human eye. Both zeaxanthin and lutein are naturally present in the macula of the human eye in the ratio 1:3. Hence, eye-care supplements must contain a combination of both these carotenoids in much the same ratio as naturally present in the human eye. Marigold flowers are rich in lutein and possess a zeaxanthin to lutein ratio about 1:16. Further, in known lutein hybrids, the content of zeaxanthin in the biomass is very low (0.1 % on dry basis). Where, although, pure lutein is plentifully available in the marigold flowers, the ratio of zeaxanthin to lutein is much less than that found in the human eye, therefore, there is a need to develop a special marigold which could overproduce zeaxanthin to ultimately formulate the most appropriate zeaxanthin-lutein blend for use in eye care supplements.

Several methods have been used for economic production of zeaxanthin substitutes; e.g. isomerisation of marigold lutein to zeaxanthin, wherein the isomerisation process produces meso-zeaxanthin instead of the 3R, 3'R isomeric form of zeaxanthin found in nature. Meso-zeaxanthin also potentially interferes with the absorption of other carotenoids (i.e. beta carotene, lutein and the dietary zeaxanthin) in the small intestine, thus, preventing the body from retaining the amount it needs of these vital carotenoids.

US Patent Nos. 6784351, 7033622 and 7575766 disclose a breed of mutant marigold which over-produces zeaxanthin. Where, the known marigold flower extract has xanthophylls 10-20 % (UV/Visible), of which, lutein is 70 - 80 % and zeaxanthin is 4 - 6 % (HPLC); the mutant marigold flower extract, thus produced, has comparatively low xanthophyll concentration (< 5%) but has much higher content of zeaxanthin (45 - 55 %) and less lutein (5-10 %). However, the marigold developed from the above disclosures has a modified biomass composition. The biomass composition tends to be waxy with a higher content of non-xanthophyll components which in turn renders downstream processing difficult. The drawbacks include: the zeaxanthin-rich biomass is difficult to dry and extract; the hydrolysis and isolation of pure zeaxanthin from the mutant marigold ester is much more complex when compared to the lutein-rich marigold extract. Therefore, it is imperative to develop a new process specially suited for the effective hydrolysis of mutant marigold extract in the endeavor to isolate pure zeaxanthin.


In nature, the xanthophylls, lutein and zeaxanthin, abundantly occur as mono and di-esters of long chain fatty acids like palmitic, stearic, myristic, oleic, linoleic, lauric and pentadecanoic acids. The lutein and zeaxanthin which are assimilated by the body in the natural ester form are biologically hydrolysed inside the human body. Only the hydrolysed free-form of lutein and zeaxanthin are transferred to the biological sites of function. High molecular weight fatty acid groups of the esters limit the dosage of free lutein and zeaxanthin consumed. Hence, the de-esterfication process is used to hydrolyze the natural carotenoid ester forms.

De-esterification of a natural carotenoid ester means a process to make free the pure carotenoid from the fatty acid backbone. There are two known processes for this purpose:

a) Ester Hydrolysis: This process is most widely used and comprises of the steps of: preparing a reaction medium - mixing the carotenoid extract (as such or by dissolving in a suitable organic medium) with an alkali metal hydroxide solution (in water or alcohol); saponification or hydrolysis reaction - carrying out liquid phase hydrolysis to free the carotenoids by cleaving the ester bond; separation of carotenoid - separation of free carotenoid powder from the reaction medium; and

b) Trans-esterification: In the hydrolysis method above, the ester bond is cleaved to free the carotenoid from the fatty acid chain. In trans-esterification, the carotenoid from the fatty acid ester is replaced by a short chain alcohol so as to precipitate the carotenoid in free-form.


Some of the disclosures discussing a process for de-esterification of carotenoid esters are listed below:

US7812198 discloses saponification of marigold extract in the presence of a quarternary ammonium base like hexa, decyl, trialkyl, ammonium hydroxide, benzyl trimethyl ammonium hydroxide in ethanol at 70 °C to 80 °C. On completion of the saponification reaction, the reaction mixture is diluted with water, crystals thus formed are filtered out, water washed and dried.

US7622599 discloses a method for the hydrolysis of carotenoid esters starting from fresh marigold flowers. The flowers are ensilaged, dehydrated, pellatised and solvent extracted to produce oleoresin. Alkali hydrolysis of the carotenoid esters in the oleoresin is done in absolute alcohol, for a period of 30 minutes at 70 - 80 °C. The xanthophylls are precipitated from the saponification mixture using ethanol-water mixture, crystals thus formed are filtered out, washed using hot water and dried.

US7629007 discloses the saponification of marigold oleoresin with aqueous alkali at 103 °C. The saponification mixture is repeatedly washed with sodium chloride solution to reduce the pH to 6.5 - 9, the resinous portion is separated, and the lipid components are removed by washing with non-polar solvent. The residue obtained is further purified by repeated washings with a mixture of water and polar organic solvents, acetone-water, methanol-water, ethanol-water, water with acetic acid and then removing the solvents by washing with water. The crystals are then filtered out and dried.

EP1911742/US7485738 discloses a method for preparation of food-grade zeaxanthin through chemical isomerization reaction from lutein. The technical issues solved by this method are: the low product yield obtained from existing methods, need of purification treatment process, and inadaptability to industrialized production. The method comprises: mixing xanthophyll crystal or its fatty acid ester with food-grade glycol or propylene glycol, for full dissolution at 60 °C to 90 °C; adding an organic alkali into the mixed liquor obtained from the above step, for isomerization reaction to take place under inertial environment; diluting the reaction solution with mixed solution of de-ionized water and ethanol; and separating the crystal so obtained by conventional separating methods; finally, vacuum drying the acquired crystals to get the zeaxanthin crystal. Glycol or propylene glycol is used in this invention for the isomerization reaction, under inertial environment, after it has fully dissolved raw material at predetermined temperatures. The product yield is reachable to more than 60 %, and the product is adaptable to industrial standards without the need for further purification treatment.

EP0834536 discloses the conversion of lutein or its naturally occurring ester into zeaxanthin by base-catalyzed isomerization achieved by heating an optionally pre-treated natural product containing lutein or pure lutein in a mixture of aqueous alkali metal hydroxide solution and either dimethyl sulphoxide or an organic solvent based on saturated aliphatic and/or aromatic hydrocarbons, at above 50 C. If an organic hydrocarbon solvent is used, the process is carried out in presence of a phase transfer catalyst.

US7271298 discloses saponification in aqueous ethanol and alkali at 40 °C to 85 °C for 3 hours to 5 hours and then neutralizing the alkali with acid water, followed by crystal isolation. The process for preparation of xanthophyll crystals containing at least 80 % of total xanthophylls with at least 90 % trans-lutein and/or zeaxanthin and trace amount of cis-lutein and other carotenoids from plant oleoresin that contains xanthophyll diesters; the process includes the steps of: a) saponifying homogeneous liquid of the plant oleoresin containing xanthophyll diesters and alcohol by adding an aqueous alkali solution at a temperature in the range of 40 °C to 85 °C for 3 hours to 5 hours; b) cooling the saponified reaction mixture to ambient temperature, and the pH is adjusted between 1 - 7; c) admixing 2 to 10 volumes of water and 0.5 to 2 volumes of alcohol per weight of the plant oleoresin, increasing the temperature between 40 °C to 85 °C, agitating slowly for a period of 0.5 to 2.0 hours, thereby forming crystalline precipitate; d) recovering xanthophyll crystals from the saponification mixture; e) washing the xanthophyll crystals 2-3 times with water at a temperature of 70 - 85 °C until supernatant is colorless; and f) leaching the xanthophyll crystals with dry ethanol and drying the xanthophyll crystals in vacuum or freeze drying the xanthophyll crystals.

US7179930 discloses a method for hydrolysis of xanthophyll esters to free xanthophylls without the use of water. The process is designed at low temperature and has a short reaction time, 45 °C and 30 minutes. This method minimizes the soap formation to facilitate the isolation of lutein crystals formed. This process deploys organic solvents like butanol, ethyl acetate, ethanol and chemicals like ion-exchange resin, quarternary ammonium salt as phase transfer catalyst, urea solution to quench the reaction and acetic acid for neutralizing the reaction mixture. The isolated lutein crystals are further stabilized by the alcohol soluble fraction obtained during the purification of the xanthophylls.

US6747177 discloses a process for the preparation of optically pure acetylenediols and their further reaction to give meso-zeaxanthin.


US6743954 discloses a process for the preparation of optically pure acetylenediols of the formulae R-I and S-I and their further reaction to give meso-zeaxanthin using class 2 and class 3 solvents like IP A, ethyl acetate, hexane, methanol and acetone.

US6743953 discloses a process for preparation of xanthophyll crystals with minimum 85 % purity of which 90 % is trans-lutein and/or zeaxanthin, using aliphatic alcoholic alkali at 65 °C to 80 °C. Distilling out the alcohol from the reaction mixture under reduced pressure, diluting the saponified crude concentrate with water and extracting the de-esterified xanthophylls to ethyl acetate, water washings to make the organic layer free of alkali and soap. Further purification of the xanthophylls is done with conventional solvent purification. This process uses aliphatic alcohols, ethyl acetate, hexane and acetone for the isolation and purification of xanthophylls.

US6504067 discloses a method for isolation and purification of xanthophylls by refining the oleoresin initially with dilute organic or inorganic acids, followed by saponification using aqueous IPA and alkali, neutralizing with acids and further purification with hexane. The method comprises cleaning of the oleoresin with acid and alkali and then subjecting to hydrolysis with aqueous alkali at 90 C for 8 hrs in the presence of emulsifiers.

US63 80442 discloses a method for isolating and purifying mixed carotenoids with higher percentage of all trans-lutein, minimizing the use of organic solvents. A solution of the oleoresin is prepared in iso-propanol at 60 °C, aqueous alkali added and stirred at 60 °C to 65 °C for 90 minutes. The saponification mixture was cooled to room temperature and diluted with de-ionized water to reduce the solvent concentration to 50 %. The mixture is allowed to stand for 1 hour and further diluted with de-ionized water and centrifuged. The crystals of xanthophylls are collected, water washed, and dried in vacuum.

US6376722 discloses a lutein to zeaxanthin isomerization process which includes a step of saponification or alkali treatment of a plant extract which contains lutein, followed by a step of heating under controlled conditions in the presence of a surfactant having a hydrophilic-lipophilic balance from 1 to 40 as a catalyst for the lutein to zeaxanthin isomerization reaction at a temperature from 70 °C to 140 °C in order to obtain a reaction product with high zeaxanthin concentrations of up to 80% with respect to total carotenoids.

US6262284 discloses simultaneous extraction and saponification of dried marigold flowers with THF (Tetrahydrofuran) and alcoholic alkali. Marigold meal was homogenized with THF and ethanolic alkali. Distilling out THF under reduced pressure after completion of saponification and washing the residue with water-alcohol mixture. The crystals were further washed with alcohol and dried.

US5998678 discloses a process for the preparation of the carotenoid zeaxanthin by isomerization and optional hydrolysis of a carotenoid precursor with an alkaline reagent in a polar organic solvent selected from the group consisting of an ether, polyhydroxyl alcohol, an ether-alcohol or a combination thereof. Also discussed is a method for improving the pigment power of an oleoresin or vegetable extract by isomerizing and optionally simultaneously saponifying the optically active carotenoids found therein by the action of an alkaline reagent in an organic medium; oxidizing the resulting optically active dihydroxycarotene and; reducing the resulting optically active keto product to generate the 3, 3' dihydroxycarotene as a racemic mixture.


US5973211 discloses a method of isomerizing lutein to yield a mixture of epimers of zeaxanthin by mixing a lutein containing extract in a glycol solution to obtain a mixture, treating the mixture with alkali to obtain an alkali containing mixture, reacting the alkali containing mixture in the presence of heat for a duration consistent with achieving a desired level of isomerization of lutein to zeaxanthin.

US5780693 discloses a process for the conversion of lutein or of its esters into zeaxanthin by base-catalyzed isomerization. The process is carried out by heating an optionally pre-treated lutein containing material in a mixture of an aqueous solution of an alkali hydroxide and either dimethyl sulphoxide or an organic solvent based on saturated aliphatic and/or aromatic hydrocarbons at temperatures in the range from about 50 °C to about 120 °C. The process is carried out in the presence of a phase transfer catalyst when an organic solvent based on hydrocarbons is used. Being carotenoids, lutein and zeaxanthin are used correspondingly, especially as pigments for egg yolk, the integuments and the subcutaneous fat of poultry, the flesh and the integuments offish and crustaceans, as well as food stuffs. This zeaxanthin is used in many applications, since in comparable dosages it produces a more intensive golden yellow pigmentation than lutein.

US5648564 discloses a method for the saponification of carotenoid diester-containing plant extract in a composition of propylene glycol and aqueous alkali to form xanthophylls crystals. Crystallization is achieved without the use of organic solvents. Process involves a) admixing the oleoresin with propylene glycol; b) adding aqueous alkali and maintaining the reaction mixture at 70 °C for around 10 hours; c) diluting with de-ionized water and centrifuge. The collected lutein crystals are further washed with hot water and vacuum dried.


US5523494 discloses a process to obtain a product with high zeaxanthin content, to be used mainly as an ingredient in poultry feed to enhance the pigmentation of boiler chickens and egg yolk, by reacting at a controlled temperature and pressure, marigold (Tagetes erecta L.) meal or its oleoresin, or formulations containing lutein, with strongly alkaline aqueous solutions under controlled conditions, to isomerize the lutein into zeaxanthin in a product with a higher pigmenting activity.

US5382714 discloses a method for isolating, purifying and recrystallizing substantially pure lutein from saponified marigold oleoresin. Lutein purified according to this method is free from many of the chemical impurities that are known constituents of marigold flowers and plant. The method involves purifying the saponified marigold oleoresin through a series of filtrations and water-alcohol washes to get crude crystals. The crystals are recrystallized form a dual solvent, an organic solvent in which lutein is highly soluble (methylene dichloride) and a second organic solvent in which lutein is only partially soluble (hexane). Upon cooling this mixture, crystals of high purity are separated, filtered and dried under vacuum. A lutein purity > 97 % with a composition > 94 % of all trans-lutein, 3 - 6 % zeaxanthin and < 3 % other carotenoids is obtained.

US20100137646 discloses a trans-esterification process to prepare xanthophylls without the use of aqueous medium. Oleoresin is first treated with a mild alkali, removing the aqueous alkali phase, and vacuum dry the oleoresin. The alkali washed oleoresin is then homogenized with an organic aprotic solvent and mixed with alkali alcoholate in alcohol. Excess alcohol is slowly added to the above mixture under reflux till the trans-esterification is complete. Aprotic solvent is distilled out and the reaction mixture cooled to room temperature. The xanthophyll crystals precipitate in the reaction medium and can be filtered out and further purified by alcohol washing.


Alkali alcoholates produced from the reaction of metals like lithium, sodium, potassium, magnesium, calcium or mixtures thereof. The alcohol selected includes branched or unbranched, substituted or unsubstitued Ci through Ci0 alcohols, exemplary alcohols include methanol, ethanol, isopropyl alcohol, butanol, tertiary butanol etc. Suitable organic aprotic solvents include methylene chloride, C5 - CJO branched or un-branched alkanes, aromatic hydrogens, etc.

US20100121112 discloses a process for the preparation of highly purified carotenoids from plant extracts, especially the marigold flower extracts. The process includes the formation, isolation and purification of carotenoids, primarily lutein and zeaxanthin, at lower temperatures with the use of at least a polar solvent and at least a non-polar solvent like pentane, hexane or heptane. The highly purified carotenoids so obtained are useful for human consumption, such as nutritional supplements and pharmaceuticals.

WO2009109712 discloses a xanthophyll composition containing trans-isomeric forms of meso-zeaxanthin, zeaxanthin and lutein useful for nutrition and health care and a process for its preparation. The process involves heating a mixture of lutein containing material along with an aromatic alcohol and alkali hydride followed by purification.

US20090238933 discloses a process for the partial synthesis of (3R,3'R)- zeaxanthin and (3R,3'S meso)-zeaxanthin from a readily accessible dehydration product of (3R,3'R,6'R)-lutein, namely, (3R)-3',4'-didehydro-beta-caroten-3-ol [(3R)-3',4'-anhydrolutein]. The process involves regioselective hydroboration of (3R)-3',4'-anhydrolutein to a mixture of (3R,3'R)-zeaxanthin and (3R, 3'S, meso)-zeaxanthin followed by separation of these carotenoids by enzyme-mediated acylation.


US2007265351 discloses a xanthophyll composition containing (trans, meso)-zeaxanthin, (trans, R, R)-zeaxanthin and (trans, R,R)-lutein useful for nutrition and health care and a process for its preparation. More particularly, the invention relates to a xanthophylls composition containing at least 80% by weight of total xanthophylls, out of which the (trans, 3R, 3'S, meso)-zeaxanthin content is at least 80%, the remaining being (trans, R, R)-zeaxanthin, (trans, R, R)-lutein and trace amounts of other carotenoids. This invention further provides a xanthophylls composition containing at least 80% by weight of total xanthophylls, out of which at least 50% being (trans, 3R, 3'S, meso)-zeaxanthin, (trans, R, R)-lutein and trace amounts of other carotenoids.

US20070161826 discloses a method of extracting free xanthophylls from the saponified marigold extract by salting out the soap from the saponification mixture, followed by purification using organic solvents-hexane, methanol, ethanol etc. to obtain free xanthophylls of 82 % purity of which 94.7 % as lutein and 5.3 % as zeaxanthin with a carotenoid recovery of 78.9 %.

EP2196207/ US7875751 describe a process for preparing lutein from marigold extract in an aliphatic hydrocarbon using alcoholic alkali hydroxide. Potassium hydroxide and sodium hydroxide are the alkali used for carrying out the process whereas the alcohol used methanol, ethanol and propan-2-ol; preferably methanol. The non polar solvent used for carrying out the process is preferably hexane. However this patent is silent on the isolation of zeaxanthin and focuses on the preparation of lutein as the major product. Also the patent discloses the steps of purification of the crystals using solvents like methanol and also the steps of additional washings using solvents like ketone for removing the residues of methanol and hexane from the final product.


US2011065805 discloses a xanthophyll composition that contains macular pigments including trans-lutein and zeaxanthin isomers, namely, (R,R)-zeaxanthin and (R,S)-zeaxanthin, derived from the plant extract/oleoresin containing xanthophylls/xanthophylls esters which is safe for human consumption and useful for nutrition and health care. The composition has at least 80 percent by weight that is total xanthophylls, of which the ratio of trans-lutein and zeaxanthin isomers being in the range of about 4:1 to about 6:1 and the ratio of the isomers of zeaxanthin being in the range of about 80 to 20:20 to 80.

US8034983 describes preparation and isolation of xanthophylls from plant sources, whereby a transesterification process is utilized without the necessity of an aqueous format.

RU2424722 discloses a method for production of vegetable oils enriched with lutein and zeaxanthin diesters involving the drying and grinding vegetable raw material. Orange-coloured marigold petals are used for production of lutein diesters and ornamental groudcherry are used for production of zeaxanthin diesters. After drying and grinding, the content of corresponding diesters is determined by a spectrophotometric method followed by mixing ground marigold petals and ground ornamental groudcherry so that lutein diesters content in relation to zeaxanthin diesters is (4-5): 1, recovering lutein and zeaxanthin diesters from the produced mixture by hot ethanol extraction, removing ethanol remains by drying and adding lutein and zeaxanthin diesters to vegetable oils in amounts sufficient for their content to be 1-10 mg per 100 ml of vegetable oil.

CN101955456 provides a method for preparing high purity xanthophyll crystal. The method includes: a) ethanol and alkali are adopted to saponify marigold extract under the conditions of heating and stirring; b) reaction product is diluted by distilled water; and c) centrifugation, washing and vacuum drying are carried out, thus obtaining xanthophyll crystal. The xanthophyll crystal is recrystallized by virtue of weak polar organic solvent and water.

Indian Patent Application No. 747/CHE/2008 relates to a process for isolation of carotenoids crystals rich in lutein from marigold flower petals, said process comprising: ensilaging said marigold flower petals under controlled anaerobic conditions; drying said ensilaged marigold flower petals to obtain a meal; extracting said meal using alcohol at a temperature in the range of about 50 °C to 60 °C to obtain oleoresin; enriching said oleoresin with alcohol at a temperature in the range of about 20 C to 32 C for about 10 to 20 minutes to obtain enriched oleoresin; treating said enriched oleoresin with alcoholic alkali at a temperature in the range of about 70 °C to 80 °C to obtain reaction mixture; adding hot water to said reaction mixture to obtain carotenoids crystals, wherein ratio of hot water to said reaction mixture is 4: 1 (v/v); filtering said carotenoids crystals; and washing and drying said carotenoids crystals from step (g) using conventional methods.

KR20090108176 describes a method for preparing a microbial composition to be added to feedstuff as a feed additive comprises the following steps of: extracting xanthophyll content including lutein from marigold bloom through alcohol fermentation for up to 24 hours; separating the extract into a solid portion and liquid portion using Kimchi lactobacillus such as Lactobacillus plantarum; concentrating the liquid portion in a concentrator and mixing the solid portion, concentrate, and excipient; drying the mixture; and adding the obtained microbial composition by 0.1-0.2 percent of the feedstuff.


US2011282083 deals with a process to convert xanthophyll esters derived from Capsicum sources to their free (non-esterified) forms. The carotenoid composition of marigold flowers and Capsicum fruits are different. The saponification medium preferably consists of hexane, potassium hydroxide and methanol. The aforementioned patent application makes use of hexane in the saponification to retain the carotenes, cryptoxanthins etc. in the hexane layer. Also to enrich the xanthophyll content of the crystals, additional step of washings by using solvents like methanol is disclosed.

ES2318992 also discloses a procedure for the preparation of essentially pure lutein from extracts of marigold.

WO11 145112 discloses a novel saponification process for the isolation and purification of highly pure carotenoids from different carotenoid rich oleoresin derived from plants and microorganisms without the use of toxic chemicals and hazardous solvents. The hydrolysis is carried out by treating the carotenoid rich oleoresin with a novel mixture of a Fatty alcohol, alkali and a fatty acid without the use of any other solvents. Further the invention explains a process of stabilizing the carotenoids from getting degraded due to high temperature and exposure time during the saponification process. Further the invention discloses an economically viable process of isolating high pure carotenoids with higher yield.

WO10125576 provides for a process for isolation of carotenoids crystals comprising drying a plant part to obtain a meal and extracting the meal with alcohol at a temperature in the range of about 50 °C to 75 °C to obtain oleoresin. The oleoresin is enriched with alcohol at a temperature in the range of about 25 °C to 50 C and hydrolyzed with alcoholic alkali at a temperature in the range of about 70 degrees centigrade to 80 degrees centigrade to obtain reaction mixture. The carotenoids crystals are precipitated from the reaction mixture by adding hot water followed by filtering, washing and drying the carotenoids crystals. The patent discloses a process for preparing crystals with lutein and zeaxanthin in required ratios.

CN101838229 provides a method for separating and purifying all-trans xanthophyll powder with high content from a Tagetes oleoresin. The method comprises the following steps of: dissolving the Tagetes oleoresin in a non-polar solvent under the heating condition; adding a phase-shift catalyst into the mixture; saponifying the mixture under the alkaline heating condition; dispersing the mixture by using a polar solvent or aqueous solution thereof; and filtering and drying the mixture under vacuum to obtain the xanthophyll powder with the total carotenoids content of over 98 % and the proportion of all-trans xanthophyll of over 92 percent.

US2010081850 describes a process for isolation of carotenoids crystals having lutein and zeaxanthin in a weight ratio of about 10:1, 5:1 or 1:1. The process comprises contacting a plant source rich in lutein with hexane and extracting at a temperature of about 40 °C to 60 °C to obtain an oleoresin rich in lutein; contacting a plant source rich in zeaxanthin with hexane and extracting at a temperature of about 40 °C to 60 C to obtain an oleoresin rich in zeaxanthin. The oleoresin obtained from both the extraction are blended and subjected to saponification reaction for the isolation of the product.

CN101486671 relates to method for preparing and stabilizing high-purity lutein ester and removing pollutants thereof; at temperature of 20 °C to 70 °C, the particles of marigold flowers are dissolved in normal butane or acetone, stirred for 0.5 h to 5 h and filtered to obtain an extraction solution; an adsorbing agent is added into the extraction solution, stirred for 0.5 h to 5 h at temperature of 20 °C to 70 °C and filtered; a mother solution is collected and dried in vacuum, thus obtaining a concentrated substance.

US2010305366 discloses a lutein extraction process comprises pulverizing marigold, adding organic solvent in the former, then placing into an ultrasonic extraction vessel for ultrasonic extraction, filtering the extracted solution, and recovering to obtain a filtrate containing lutein ester; adding an alkaline solution that is 2 times of the filtrate volume in the filtrate recovered in the above step, and performing saponification on the lutein ester in the filtrate at 50 °C to 70 °C; washing the resultant after saponification with de-ionized water until lutein crystallization occurs, centrifuging the solution containing the lutein crystals to obtain crude crystals of lutein; subjecting the crude crystals of lutein to recrystallization; washing the lutein crystals obtained by recrystallization and drying to obtain lutein crystal product.

CN101429146 provides a high-purity lutein crystal which can be stably prepared from marigold petals in large scale, as well as a production method thereof. The production method comprises the following steps: alkane is adopted as a solvent to extract lutein diester in the marigold petals; an extract is decompressed, concentrated, subjected to saponification in a system consisting of ethanol and NaOH and mixed with water so as to obtain a coarse lutein crystal; and a coarse crystal product is recrystallized by use of halogenated alkane, mixed with ethanol, subjected to high vacuum distillation so as to remove the halogenated alkane, filtered, decompressed and dried, so as to obtain the lutein crystal the purity of which is up to over 90 %.

Indian patent application No.l322/MUM/2008 provides a xanthophyll composition containing at least 80 percent by weight of total xanthophylls,(out) of which the trans- lutein content is at least 80 % by weight and the remaining being zeaxanthin isomers, namely,: R,R-zeaxanthin and R,S - zeaxanthin and traces of other carotenoids derived from the plant extract. Oleoresin containing xanthophyll esters which is safe for human consumption and useful for nutrition and health care. The invention also provides a process for the preparation of the composition.

The processes listed in the prior art above are suitable for the de-esterification of marigold carotenoids and are typically designed for the lutein-rich marigold extracts which are different in composition and behavior when compared to the zeaxanthin rich marigold mutant extracts. Further to enrich the concentration of the carotenoids in the final product additional steps of purification is done which is time consuming and adds to the overall cost thus making the process costly. Mutant marigold flowers rich in zeaxanthin have a modified biomass composition with a higher content of non-xanthophyll components. This renders the biomass waxy, making its processing/isolation difficult and complex in comparison with the known high lutein marigold extracts. Therefore, there is felt a need to provide a process for the isolation of free zeaxanthin from mutant marigold extracts that facilitates optimum conversion of zeaxanthin esters to free zeaxanthin and easy recovery of zeaxanthin crystals from the reaction mixture.

OBJECTS

Some of the objects of the present disclosure are as follows:

An object of the present disclosure is to provide a process for the isolation of free zeaxanthin from mutant marigold extracts.


Another object of the present disclosure is to provide a process for the isolation of free zeaxanthin from mutant marigold extracts which facilitates optimum conversion of zeaxanthin esters to free zeaxanthin.

Yet another object of the present disclosure is to provide a process for the isolation of free zeaxanthin from mutant marigold extracts where the zeaxanthin crystals are easily isolated from the reaction mixture containing the un-hydrolyzed components and free carotenes.

Still another object of the present disclosure is to provide a process for the isolation of free zeaxanthin from mutant marigold extracts which provides an improved zeaxanthin recovery.

One more object of the present disclosure is to provide a process for the isolation of free zeaxanthin from mutant marigold extracts using a well accepted food-grade solvent.

SUMMARY

In accordance with the present disclosure, there is provided a process for the isolation of free zeaxanthin from a marigold extract prepared from mutant marigold plant, said process comprising the following steps:

(a) homogenizing the marigold extract for a time period in the range of 10 minutes to 20 minutes under stirring at a temperature ranging between 50 °C and 60 °C to obtain a homogenized marigold extract;


(b) mixing the homogenized marigold extract with hydrocarbon solvent to form a uniform mixture;

(c) slowly adding an alcoholic alkali hydroxide solution to the uniform mixture of step (b) with stirring at a temperature ranging between 50 °C and 60 °C for a time period in the range of 20 minutes to 45 minutes and continuing stirring after addition of alcoholic alkali hydroxide at a temperature in the range of 60°C to 90 °C for a time period between 60 minutes and 95 minutes to obtain a reaction mixture;

(d) analyzing the reaction mixture for completion of de-esterification by HPLC followed by removal of alcohol from the reaction mixture by distillation to obtain a de-esterified substantially alcohol free mixture;

(e) diluting the de-esterified substantially alcohol free mixture at a temperature in the range of 50 °C to 70 °C with water and cooling at a temperature ranging between 10 °C and 15 °C after dilution to obtain a biphasic mixture comprising an aqueous layer and a hydrocarbon layer;

(f) separating the hydrocarbon layer from the aqueous layer of the biphasic mixture of step (e) followed by the centrifugation of the aqueous layer to obtain crystals; and

(g) washing the crystals obtained after centrifugation with water followed by vacuum drying to obtain the final product.

Preferably, the plant extract is prepared from mutant marigold flowers.

Typically, the hydrocarbon solvent is at least one selected from the group of alkanes having carbon atoms greater than 25.

Preferably, the hydrocarbon solvent is liquid paraffin.


Typically, the alcohol is at least one selected from the group comprising of methanol, ethanol, propanol, propan-2-ol and 1- butanol.

Preferably, the alcohol is ethanol.

Typically, the alkali hydroxide is at least one selected from the group comprising of lithium hydroxide, sodium hydroxide potassium hydroxide and cesium hydroxide.

Preferably, the alkali hydroxide is potassium hydroxide.

Typically, the percentage of alkali hydroxide present in the alcohol is in the range of 15% to 30%.

In accordance with the process of the present disclosure, the method step (f) optionally involves diluting the separated aqueous layer with water before centrifugation.

Typically, the vacuum drying of the product obtained is carried out at a temperature ranging between 40 °C and 60 °C for a time period in the range of 1 hour to 5 hours.

The product comprising zeaxanthin crystals prepared by the process of the present disclosure has moisture content less than 3%.

Typically, the percentage of xanthophyll concentration in the product is between 45% and 72%.


The product formed in accordance with the process of the present disclosure has carotenoid profile of 85% to 95% of zeaxanthin.

Typically, the percentage of zeaxanthin recovery from the input mutant marigold extract is in the range of 55 % to 70%.

In accordance with the present disclosure there is provided a product comprising zeaxanthin crystals obtained by the process of the present disclosure.

DETAILED DESCRIPTION

The present disclosure envisages a process for the isolation of free zeaxanthin from a marigold extract prepared from mutant marigold plant, preferably from its flowers having zeaxanthin content in the range of 0.2 to 0.25 % based on the dried mass of the flowers. Mutant marigold flowers rich in zeaxanthin have a modified biomass composition with a higher content of non-xanthophyll components. This renders the biomass waxy, making its processing/isolation difficult and complex in comparison with the known high lutein marigold extracts. In the zeaxanthin-rich marigold extract conventional carotenoid extraction and isolation processes are cumbersome, ultimately resulting in inferior zeaxanthin recovery. Therefore, there is a need for an isolation/de-esterification process for the hydrolysis of the mutant marigold zeaxanthin extract especially suitable for zeaxanthin-rich mutant marigold extracts.

In accordance with the present disclosure, there is provided a process for the isolation of free zeaxanthin from a marigold extract prepared from mutant marigold plant; preferably from its flowers, said process comprising the following steps:


(a) homogenizing the marigold extract for a time period in the range of 10 minutes to 20 minutes under stirring at a temperature ranging between 50 °C and 60 °C to obtain a homogenized marigold extract;

(b) mixing the homogenized marigold extract with hydrocarbon solvent to form a uniform mixture;

(c) slowly adding an alcoholic alkali hydroxide solution to the uniform mixture of step (b) with stirring at a temperature ranging between 50 °C and 60 °C for a time period in the range of 20 minutes to 45 minutes and continuing stirring after addition of alcoholic alkali hydroxide at a temperature in the range of 60 C to 90 C for a time period between 60 minutes and 95 minutes to obtain a reaction mixture;

(d) analyzing the reaction mixture for completion of de-esterification by HPLC followed by removal of alcohol from the reaction mixture by distillation to obtain a de-esterified substantially alcohol free mixture;

(e) diluting the de-esterified substantially alcohol free mixture at a temperature in the range of 50 °C to 70 °C with water and cooling at a temperature ranging between 10 °C and 15 °C after dilution to obtain a biphasic mixture comprising an aqueous layer and a hydrocarbon layer;

(f) separating the hydrocarbon layer from the aqueous layer of the biphasic mixture of step (e) followed by the centrifugation of the aqueous layer to obtain crystals; and

(g) washing the crystals obtained after centrifugation with water followed by vacuum drying to obtain the final product.

The process of the present disclosure uses an effective organic medium that freely dissolves the zeaxanthin-rich marigold extract to give a low viscous homogenous solution to facilitate efficient hydrolysis. Further, the organic medium is adapted to facilitate high temperature hydrolysis in a short duration to dissolve the normally un-hydrolyzed components in the extract thereby facilitating easy crystal formation and isolation of the zeaxanthin.

The organic medium that is used in the process of the present disclosure is hydrocarbon solvent that is at least one selected from the group of alkanes having carbon atoms greater than 25.

Preferably, the hydrocarbon solvent used in the process of the present disclosure is liquid paraffin. Liquid paraffin is a free flowing liquid which allows thorough mixing of the reactants and can be heated to higher temperatures.

In accordance with the process of the present disclosure, there is provided a hydrolysis medium consisting of the liquid paraffin and alcoholic alkali hydroxide for optimum conversion of the zeaxanthin esters present in the mutant marigold extract to free zeaxanthin.

Typically, the alcohol used for the hydrolysis in the process of the present disclosure is at least one selected from the group comprising of methanol, ethanol, propanol, propan-2-ol and 1-butanol.

Preferably, the alcohol used is ethanol.

The alkali hydroxide used for the de-esterification reaction in accordance with the present disclosure is at least one selected from the group comprising of lithium hydroxide, sodium hydroxide, potassium hydroxide and cesium hydroxide.


Preferably, the alkali hydroxide used is potassium hydroxide.

Typically, the process of the present disclosure uses a hydrolysis medium consisting of a mixture of liquid paraffin and ethanolic potassium hydroxide. In accordance with the process of the present disclosure, the alcoholic alkali hydroxide solution is slowly added to the uniform mixture of mutant marigold extract with stirring at a temperature in the range of 50 °C to 60 °C for duration of 20 minutes to 45 minutes. The stirring is continued after addition of alcoholic alkali hydroxide at a temperature in the range of 60 °C to 90 °C for a time period between 60 minutes and 95 minutes.

The analysis of the reaction mixture for completion of de-esterification is done by HPLC in the present disclosure. Other known methods can also be used for analyzing the completion of reaction.

After the completion of reaction, the alcohol from the reaction mixture is removed by distillation under reduced pressure. Preferably, ethanol is distilled at a temperature in the range of 50 °C to 60 °C under reduced pressure. The de-esterified substantially alcohol free mixture is then diluted with water at temperature ranging between 50 °C and 70 °C with continuous stirring. The diluted reaction mixture is then cooled to a temperature in the range of 10 °C to 15 °C to form a biphasic mixture. The supernatant paraffin layer is separated and the aqueous layer is subjected to centrifugation to obtain the product crystals. The non-hydrolyzed components of the extract and free carotenes (like beta carotene) are efficiently solubilized and isolated along with the liquid paraffin and zeaxanthin crystals that are formed remains in the aqueous layer and are easily separable by centrifugation. The aqueous layer obtained after separation is optionally diluted with water before centrifugation.


The centrifugation is done at 12000 rpm to 15000 rpm to obtain crystals of the product.

The product formed in accordance with the present disclosure is further subjected to washings with water to remove excess alkali and potassium salts of fatty acids. Preferably, de-ionized water is used for washing the crystals.

After washing, the product obtained is finally subjected to drying. Drying can be done by any known methods. Drying of the crystals obtained by the process of the present disclosure is done in vacuum drier at a temperature ranging between 40 °C and 60 °C for a time period in the range of 1 hour to 5 hours. The final product obtained by the process of the present disclosure has moisture content of less than 3%.

ANALYSIS OF THE PRODUCT;

The product obtained by the process of the present disclosure has xanthophyll concentration in the range of 45% to 72% as measured by UV- Visible spectrometry. The carotenoid profile by HPLC analysis of the product formed shows 85% to 95% of zeaxanthin and 4.5 % to 7.5% lutein.

The percentage of zeaxanthin recovery from the input marigold extract is in the range of55%to70%.

TEST RESULTS

The disclosure will now be described with respect to the following examples which do not limit the scope and ambit of the disclosure in anyway and only exemplify the disclosure.


EXAMPLE 1:

500 g of mutant marigold extract, containing 2.95 % xanthophylls and 1.33 % zeaxanthin (45 % of the total xanthophylls content) was taken in a hot water jacketed reaction vessel with stirrer. The extract was homogenized for 15 minutes under stirring at 56 °C.

500 g liquid paraffin was slowly added to the extract under stirring over a period of 10 minutes to get a uniform solution. Separately, 250 g of potassium hydroxide was dissolved in 1000 ml of 95 % ethyl alcohol to obtain ethanolic alkali solution. The ethanolic alkali solution was added to the extract solution with continuous stirring at 56 °C over a period of 20 minutes. The stirring was continued at 70 °C to 75 °C for 70 minutes. After ensuring the completion of de-esterification by HPLC analysis, ethanol was distilled off at 55 °C under reduced pressure.500 ml de-ionized water was added to the stirred reaction mixture at 70 °C along with further stirring. The reaction mixture was then cooled to 10 °C. The supernatant paraffin layer was separated. The aqueous layer was further diluted with 15 Litres of de-ionized water and mixed well. The mixture was centrifuged at 15000 rpm. The crystals were collected and washed with de-ionized water to remove excess alkali and potassium salts of fatty acids. The crystals were finally vacuum dried at 50 °C.

7.43 g of zeaxanthin crystals with 65.01 % xanthophylls were obtained. Carotenoid profile of the crystals (measured by HPLC) showed 90 % zeaxanthin and 6.8 % lutein. The zeaxanthin recovery from input extract was 65.4 %.

EXAMPLE 2:

500 g of mutant marigold extract, containing 3.856 % xanthophylls and 1.877 % zeaxanthin (48.68% of the total xanthophylls content) was taken in a hot water jacketed reaction vessel with stirrer. The extract was homogenized for 15 minutes under stirring at 56 °C.

250 g liquid paraffin was slowly added to the extract under stirring over a period of 10 minutes to get a uniform solution. Separately, 200 g of potassium hydroxide was dissolved in 800 ml of 95 % ethyl alcohol to obtain ethanolic alkali solution. The ethanolic alkali solution was added to the extract solution with continuous stirring at 56 °C over a period of 20 minutes. The stirring was continued at 70 °C to 75 °C for 60 minutes. After ensuring the completion of de-esterification by HPLC analysis, ethanol was distilled off at 55 °C under reduced pressure. 500 ml de-ionized water was added to the stirred reaction mixture at 70 °C along with further stirring. The reaction mixture was then cooled to 10 °C. The supernatant paraffin layer was separated. The aqueous layer was further diluted with 12 litres of de-ionized water and mixed well. The mixture was centrifuged at 15000 rpm. The crystals were collected and washed with de-ionized water to remove excess alkali.
The crystals were finally vacuum dried at 50 °C.

9.52 g of zeaxanthin crystals with 68.5 % xanthophylls content were obtained. Carotenoid profile of the crystals (measured by HPLC) was 89.2 % zeaxanthin and 7.01 % lutein. The zeaxanthin recovery from input marigold extract was 62 %.

EXAMPLE 3:

500 g of mutant marigold extract, containing 4.33 % xanthophylls and 2.25 % zeaxanthin (52.24 % of the total xanthophylls), was taken in a hot water jacketed reaction vessel with stirrer. The extract was homogenized for 15 minutes under stirring at 56 °C.
100 g liquid paraffin was slowly added to the extract under stirring over a period of 10 minutes to get a uniform solution.. Separately, 250 g of potassium hydroxide was dissolved in 1000 ml of 95 % ethyl alcohol to obtain ethanolic alkali solution. The ethanolic alkali solution was added to the extract solution with continuous stirring at 56 °C over a period of 30 minutes. The stirring was further continued at 70 °C to 75 °C for 90 minutes. . After ensuring the completion of de-esterification by HPLC analysis, ethanol was distilled off at 55 °C under reduced pressure. 500 ml de-ionized water was added to the reaction mixture at 70 °C along with further stirring. The reaction mixture was cooled to 10 °C. The supernatant paraffin layer was separated. The aqueous layer was further diluted with 15 litres of de-ionized water and mixed well. The mixture was centrifuged at 15000 rpm. The crystals were collected and washed with de-ionized water to remove excess alkali and potassium salts of fatty acids. The crystals were finally vacuum dried at 50 °C.

10.74 g of zeaxanthin crystals with 70 % xanthophylls content were obtained. Carotenoid profile of the crystals (measured by HPLC) was 92 % zeaxanthin and 6.2 % lutein. The zeaxanthin recovery from input extract was 61.5%.

EXAMPLE 4

2 kg of mutant marigold extract containing 22.72g/kg of xanthophylls and 10.7g/kg zeaxanthin (47.16% of total xanthophylls) was taken in a hot water jacketed reaction vessel with stirrer. The extract was homogenized for 15 minutes under stirring at 56 °C.
0.2 kg of liquid paraffin was slowly added to the extract under stirring over a period of 10 minutes to get a uniform solution. 25% ethanolic alkali was prepared by dissolving 0.5 kg of potassium hydroxide in 2 litres of ethanol of 95% purity. The ethanolic alkali solution was slowly added to the reaction vessel containing marigold extract and liquid paraffin with continuous stirring at 56 C over a period of 15 minutes. The stirring was continued at 65 C for 60 minutes. After ensuring the completion of de-esterification by HPLC analysis, ethanol was distilled off at 55 °C under reduced pressure. The reaction mixture was diluted with 50 litres of de-ionized water, maintained at a temperature of 70 °C with continuous stirring. The diluted reaction mixture was then cooled to 10 °C. Supernatant paraffin layer was separated. The aqueous layer was centrifuged at 13000 rpm. The crystals were collected and washed with de-ionized water to remove excess alkali and potassium salts of fatty acids. The wet crystals obtained were finally dried in vacuum drier at 50 °C for 1 hour to a moisture content < 3%.

The yield of the final zeaxanthin crystal was 1.5 % (30 g) with xanthophyll concentration 45.5%, as analyzed by UV-Visible spectrophotometer. The carotenoid profile by HPLC analysis was 89.82% zeaxanthin, 6.01% lutein, 0.36% betacarotene and 0.39% cryptoxanthin. Moisture content of the crystals was 2.7%.

Zeaxanthin recovery of the final crystals from the input marigold extract was 57.3%.

EXAMPLE 5

5 kg of mutant marigold extract containing 38.6g/kg of xanthophylls and 18.8 g/kg zeaxanthin (48.68% of total xanthophylls), was taken in a hot water jacketed reaction vessel, with stirrer. The extract was homogenized for 15 minutes under stirring at 56 °C.

2.5 kg of liquid paraffin was slowly added to the extract under stirring over a period of 10 minutes to get a uniform solution. 25% ethanolic alkali was prepared by dissolving 1.25 kg of potassium hydroxide in 5 litres of ethanol of 95% purity. The ethanolic alkali solution was slowly added to the reaction vessel containing marigold extract and liquid paraffin with continuous stirring at 56 C. over a period of 15 minutes. The stirring was continued at 70 °C for 60 minutes. After ensuring the completion of de-esterification by HPLC analysis, ethanol was distilled off at 55 °C under reduced pressure. The reaction mixture was diluted with 125 litres of de-ionized water maintained at a temperature of 70 °C, with continuous stirring. The diluted reaction mixture was cooled to 10 °C. Supernatant paraffin layer was separated. The aqueous layer was then centrifuged at 13000 rpm. The crystals were collected and washed with de-ionized water to remove excess alkali and potassium salts of fatty acids. The wet crystals obtained were finally dried in vacuum drier at 50 C for 2 hours to a moisture content < 3%.

The yield of the final zeaxanthin crystal was 2.04 % (102 g) with xanthophylls concentration 61.68%, as analyzed by UV- Visible spectrophotometer. The carotenoid profile by HPLC analysis was 89.03% zeaxanthin, 6.94% lutein, 0.9% betacarotene and 0.87% cryptoxanthin. Moisture content of the crystals was 2.2 %.

Zeaxanthin recovery of the final crystals from the input marigold extract was 59.59 %

EXAMPLE 6

10 kg of mutant marigold extract containing 38.6 g/kg of xanthophylls and 1.88 % zeaxanthin (48.68% of total xanthophylls) was taken in a hot water jacketed reaction vessel, with stirrer. The extract was homogenized for 15 minutes under stirring at 56 °C.

10 kg of liquid paraffin was slowly added to the extract under stirring over a period of 15 minutes to get a uniform solution. 20% ethanolic alkali was prepared by dissolving 2 kg of potassium hydroxide in 10 litres of ethanol of 95% purity. The ethanolic alkali solution was slowly added to the reaction vessel containing marigold extract and liquid paraffin with continuous stirring at 56 °C over a period of 30 minutes. The stirring was continued at 70 °C for 80 minutes. After ensuring the completion of de-esterification by HPLC analysis, ethanol was distilled off at 55 °C under reduced pressure. The reaction mixture was diluted with 270 litres of de-ionized water maintained at a temperature of 70 °C, with continuous stirring. The diluted reaction mixture was cooled to 10 °C. Supernatant paraffin layer was separated. The aqueous layer was then centrifuged at 13000 rpm. The crystals were collected and washed with de-ionized water to remove excess alkali and potassium salts of fatty acids. The wet crystals obtained were finally dried in vacuum drier at 50 °C for 3 hours to a moisture content < 3%.

The yield of the final zeaxanthin crystal was 1.98 %( 198 g) with xanthophyll concentration 62.23 %, as analyzed by UV-Visible spectrophotometer. The carotenoid profile by HPLC analysis was 91.13% zeaxanthin, 4.73% lutein, 0.12% betacarotene and 0.28% cryptoxanthin. Moisture content of the crystals was 2.32%.

Zeaxanthin recovery of the final crystals from the input marigold extract was 59.72 %.

EXAMPLE 7

5 kg of mutant marigold extract containing 35.7g /kg of xanthophylls and 19.56g/kg zeaxanthin (54.79% of total xanthophylls) was taken in a hot water jacketed reaction vessel, with stirrer. The extract was homogenized for 15 minutes under stirring at 2.5 kg of liquid paraffin was slowly added to the extract under stirring over a period of 10 minutes to get a uniform solution. 25% ethanolic alkali was prepared by dissolving 1.0 kg of potassium hydroxide in 4 litres of ethanol of 90% purity. The ethanolic alkali solution was slowly added to the reaction vessel containing marigold extract and liquid paraffin with continuous stirring at 56 °C over a period of 15 minutes. The stirring was continued at 70 °C for 90 minutes. After ensuring the completion of de-esterification by HPLC analysis, ethanol was distilled off at 55 °C under reduced pressure. The reaction mixture was diluted with 135 litres of de-ionized water maintained at a temperature of 70 °C, with continuous stirring. The diluted reaction mixture was cooled to 10 °C. Supernatant paraffin layer was separated. The aqueous layer was then centrifuged at 13000 rpm. The crystals were collected and washed with de-ionized water to remove excess alkali and potassium salts of fatty acids. The wet crystals obtained were finally dried in vacuum drier at 50 °C for 2 hours to a moisture content < 3%.

The yield of the final zeaxanthin crystal was 1.86 %( 93 g) with xanthophyll concentration 71.8%, as analyzed by UV-Visible spectrophotometer. The carotenoid profile by HPLC analysis was 91.12 % zeaxanthin, 4.63% lutein, 0.11% betacarotene and 0.27 % cryptoxanthin. Moisture content of the crystals was 2.2%.

Zeaxanthin recovery of the final crystals from the input marigold extract was 62.23 %.

EXAMPLE 8

10 kg of mutant marigold extract containing 35.7 g/kg of xanthophylls and 19.56 g/kg zeaxanthin (54.79% of total xanthophylls) was taken in a hot water jacketed reaction vessel, with stirrer. The extract was homogenized for 15 minutes under stirring at 56 °C.

5 Kg of liquid paraffin was slowly added to the extract under stirring over a period of 10 minutes to get a uniform solution. 25% ethanolic alkali was prepared by dissolving 2.0 kg of potassium hydroxide in 8 litres of ethanol of 90% purity. The ethanolic alkali solution was slowly added to the reaction vessel containing marigold extract and liquid paraffin with continuous stirring at 56 °C over a period of 20 minutes. The stirring was continued at 65 °C for 90 minutes. After ensuring the completion of de-esterification by HPLC analysis, ethanol was distilled off at 55 °C under reduced pressure. The reaction mixture was diluted with 270 Litres of de-ionized water maintained at a temperature of 70 °C, with continuous stirring. The diluted reaction mixture was cooled to 10 °C. Supernatant paraffin layer was separated. The aqueous layer was then centrifuged at 13000 rpm. The crystals were collected and washed with de-ionized water to remove excess alkali and potassium salts of fatty acids. The wet crystals obtained were finally dried in vacuum drier at 50 °C for 3 hours to a moisture content < 3%.

The yield of the final zeaxanthin crystal was 1.9 % (190 g) with xanthophylls concentration 70.56 %, as analyzed by UV-Visible spectrophotometer. The carotenoid profile by HPLC analysis was 91.1% zeaxanthin, 4.75% lutein, 0.12% betacarotene and 0.29% cryptoxanthin. Moisture content of the crystals 2.65%.

Zeaxanthin recovery of the final crystals from the input marigold extract was 62.43 %


EXAMPLE 9

10 kg of mutant marigold extract containing 28.8 /kg of xanthophylls d 16.19g/kg zeaxanthin (56.22% of total xanthophylls) was taken in a hot water jacketed reaction vessel, with stirrer. The extract was homogenized for 15 minutes under stirring at 56 °C.
5 kg of liquid paraffin was slowly added to the extract under stirring over a period of 10 minutes to get a uniform solution. 25% ethanolic alkali was prepared by dissolving 2.5 kg of potassium hydroxide in 10 litres of ethanol of 90% purity. The ethanolic alkali solution was slowly added to the reaction vessel containing marigold extract and liquid paraffin with continuous stirring at 56 °C over a period of 40 minutes. The stirring was continued at 75 °C for 90 minutes. After ensuring the completion of de-esterification by HPLC analysis, ethanol was distilled off at 55 °C under reduced pressure. The reaction mixture was diluted with 270 litres of de-ionized water maintained at a temperature of 70 °C, with continuous stirring and cooled to 10 °C. Supernatant paraffin layer was separated. The aqueous layer was then centrifuged at 13000 rpm. The crystals were collected and washed with de-ionized water to remove excess alkali and potassium salts of fatty acids. The wet crystals obtained were finally dried in vacuum drier at 50 °C for 3 hours to a moisture content < 3%. The yield of the final zeaxanthin crystal was 1.96 %( 196 g) with xanthophyll concentration 54.84%, as analyzed by UV-Visible spectrophotometer. The carotenoid profile by HPLC analysis was 89.03% zeaxanthin, 6.94% lutein, 0.9% betacarotene and 0.87% cryptoxanthin. Moisture content of the crystals 2.75%.

Zeaxanthin recovery of the final crystals from the input marigold extract was 59.10 %


EXAMPLE 10

10 kg of mutant marigold extract containing 28.8 g/kg of xanthophylls and 16.19 g/kg zeaxanthin (56.22% of total xanthophylls) was taken in a hot water jacketed reaction vessel, with stirrer. The extract was homogenized for 15 minutes under stirring at 56 °C.

1 kg of liquid paraffin was slowly added to the extract under stirring over a period of 10 minutes to get a uniform solution. 25% ethanolic alkali was prepared by dissolving 2.5 kg of potassium hydroxide in 10 litres of ethanol of 90% purity. The ethanolic alkali solution was slowly added to the reaction vessel containing marigold extract and liquid paraffin with continuous stirring at 56 °C over a period of 30 minutes. The stirring was continued at 75 °C for 90 minutes. After ensuring the completion of de-esterification by HPLC analysis, ethanol was distilled off at 55 C under reduced pressure. The reaction mixture was diluted with 270 litres of de-ionized water maintained at a temperature of 70 °C, with continuous stirring and cooled to 10 C. Supernatant paraffin layer was separated. The aqueous layer was then centrifuged at 13000 rpm. The crystals were collected and washed with de-ionized water to remove excess alkali and potassium salts of fatty acids. The wet crystals obtained were finally dried in vacuum drier at 50 °C for 3 hours to a moisture content < 3%.

The yield of the final zeaxanthin crystal was 2.09 %( 209 g) with xanthophylls concentration 50.24%, as analyzed by UV-Visible spectrophotometer. The carotenoid profile by HPLC analysis was 88.04% zeaxanthin, 7.26% lutein, 0.8% betacarotene and 1.2% cryptoxanthin. Moisture content of the crystals was 2.85%.

Zeaxanthin recovery of the final crystals from the input marigold extract was 56.97 %


EXAMPLE 11

10 kg of mutant marigold extract containing 30.9 g/kg of xanthophylls and 15 g/kg zeaxanthin (48.6% of total xanthophylls) was taken in a hot water jacketed reaction vessel, with stirrer. The extract was homogenized for 15 minutes under stirring at 56 °C.
5 kg of liquid paraffin was slowly added to the extract under stirring over a period of 10 minutes to get a uniform solution. 25% ethanolic alkali was prepared by dissolving 2.5 kg of potassium hydroxide in 10 litres of ethanol of 90% purity. The ethanolic alkali solution was slowly added to the reaction vessel containing marigold extract and liquid paraffin with continuous stirring at 56 °C over a period of 30 minutes. The stirring was continued at 75 °C for 90 minutes. After ensuring the completion of de-esterification by HPLC analysis, ethanol was distilled off at 55 °C under reduced pressure. The reaction mixture was diluted with 270 litres of de-ionized water maintained at a temperature of 70 °C, with continuous stirring and cooled to 10 °C. Supernatant paraffin layer was separated. The aqueous layer was then centrifuged at 13000 rpm. The crystals were collected and washed with de-ionized water to remove excess alkali and potassium salts of fatty acids. The wet crystals obtained were finally dried in vacuum drier at 50 °C for 3 hours to a moisture content < 3%. The yield of the final zeaxanthin crystal was 1.86 %( 180 g) with xanthophyll concentration 54.84%, as analyzed by UV-Visible spectrophotometer. The carotenoid profile by HPLC analysis was 88.04% zeaxanthin, 7.26 % lutein, 0.8% betacarotene and 1.2 % cryptoxanthin. Moisture content of the crystals 2.65%.

Zeaxanthin recovery of the final crystals from the input marigold extract was 59.86 %.


EXAMPLE 12

10 kg of Marigold Extract containing 30.9 g/kg of xanthophylls and 15g/kg zeaxanthin (48.6% of total xanthophylls) was taken in a hot water jacketed reaction vessel, with stirrer. The extract was homogenized for 15 minutes under stirring at 56 °C.

5 kg of liquid paraffin was slowly added to the extract under stirring over a period of 10 minutes to get a uniform solution. 20% ethanolic alkali was prepared by dissolving 2 kg of potassium hydroxide in 10 litres of ethanol of 90% purity. The ethanolic alkali solution was slowly added to the reaction vessel containing marigold extract and liquid paraffin with continuous stirring at 56 °C over a period of 30 minutes. The stirring was continued at 70 °C for 60 minutes. After ensuring the completion of de-esterification by HPLC analysis, ethanol was distilled off at 55 °C under reduced pressure. The reaction mixture was diluted with 270 litres of demineralised water maintained at a temperature of 70 °C, with continuous stirring. The diluted reaction mixture was cooled to 10 °C. Supernatant paraffin layer was separated. The aqueous layer was then centrifuged at 13000 rpm. The crystals were collected and washed with de-ionized water to remove excess alkali and potassium salts of fatty acids. The wet crystals obtained were finally dried in vacuum drier at 50 C for 3 hours to a moisture content < 3%.

The yield of the final zeaxanthin crystal was 2 %( 200 g) with xanthophyll concentration 48.5%, as analyzed by UV-Visible spectrophotometer. The carotenoid profile by HPLC analysis was 91.13% zeaxanthin, 4.73% lutein, 0.12% betacarotene and 0.28% cryptoxanthin. Moisture content of the crystals was 2.5%.

Zeaxanthin recovery of the final crystals from the input marigold extract was 58.9 %


EXAMPLE 13

10 kg of mutant marigold extract containing 30.9 g/kg of xanthophylls and 15g/kg zeaxanthin (48.6% of total xanthophylls) was taken in a hot water jacketed reaction vessel, with stirrer. The extract was homogenized for 15 minutes under stirring at 56 °C.

10 kg of liquid paraffin was slowly added to the extract under stirring over a period of 10 minutes to get a uniform solution. 25% ethanolic alkali was prepared by dissolving 2.5 kg of potassium hydroxide in 10 litres of ethanol of 90% purity. The ethanolic alkali solution was slowly added to the reaction vessel containing marigold extract and liquid paraffin with continuous stirring at 56 °C over a period of 30 minutes. The stirring was continued at 75 °C for 80 minutes. After ensuring the completion of de-esterification by HPLC analysis, ethanol was distilled off at 55 °C under reduced pressure. The reaction mixture was diluted with 270 litres of demineralised water maintained at a temperature of 70 °C, with continuous stirring and cooled to 10 °C. Supernatant paraffin layer was separated. The aqueous layer was then centrifuged at 13000 rpm. The crystals were collected and washed with de-ionized water to remove excess alkali and potassium salts of fatty acids. The wet crystals obtained were finally dried in vacuum drier at 50 °C for 3 hours to a moisture content < 3%.

The yield of the final zeaxanthin crystal was 1.89 %( 189 g) with xanthophyll concentration 52.29%, as analyzed by UV-Visible spectrophotometer. The carotenoid profile by HPLC analysis was 91.13% zeaxanthin, 4.73% lutein, 0.9% betacarotene and 0.87% cryptoxanthin. Moisture content of the crystals was 2.32%.

Zeaxanthin recovery of the final crystals from the input marigold extract was 60 %.


TECHNICAL ADVANTAGES

A process for the isolation of free zeaxanthin from mutant marigold extracts, as disclosed in the present disclosure has several technical advantages including but not limited to the realization of:

• the process of the present disclosure uses well accepted food-grade solvent-liquid paraffin; the use of liquid paraffin is safe for consumption and finds wide acceptance in food, cosmetics and pharmaceutical applications whereas the conventional organic solvents like methanol, acetone, isopropyl alcohol, ethyl acetate, hexane, etc. has an increasing uncertainity of food safety;

• the process of the present disclosure provides optimum conversion of zeaxanthin esters to free zeaxanthin by using liquid paraffin which reduces the viscosity of the marigold extract to ensure a high level of homogeneity in the reaction mixture; further, high boiling point of the liquid paraffin and low viscosity of the reacting medium ensures a smooth reaction;

• the process of the present disclosure gives zeaxanthin crystals which are facilitated and easily isolated from the reaction mixture due to the low viscosity of the reaction mixture, where the un-hydrolyzed components of the mutant marigold extracts and free carotenes like beta-carotene remain dissolved in the liquid paraffin easing the separation of the zeaxanthin crystals;

• the process of the present disclosure provides an improved zeaxanthin recovery, where, due to the high boiling point of the liquid paraffin and low viscosity of the reacting medium the duration of the saponification process is abridged which results in minimal xanthophyll degradation and improved zeaxanthin recovery;

• the present disclosure provides an easy process for the manufacturing of zeaxanthin crystals from mutant marigold extract which is comparatively less time consuming and can be carried out from small scale to large scale; and

• the present disclosure provides a simple, safe and cost effective process for the manufacturing of zeaxanthin crystals from mutant marigold extract.

The product comprising zeaxanthin crystals obtained by the process of the present disclosure has non limited applications including its use in eye care supplements, nutritional supplements and pharmaceutical applications.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

The use of the expression "at least" or "at least one" suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the invention to achieve one or more of the desired objects or results.

Any discussion of documents, acts, materials, devices, articles or the like that has been included in this specification is solely for the purpose of providing a context for the invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the invention as it existed anywhere before the priority date of this application.

The numerical values mentioned for the various physical parameters, dimensions or quantities are only approximations and it is envisaged that the values higher/lower than the numerical values assigned to the parameters, dimensions or quantities fall within the scope of the invention, unless there is a statement in the specification specific to the contrary.

The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.


Claim:

1. A process for the isolation of free zeaxanthin from a marigold extract prepared from mutant marigold plant, said process comprising the following steps:

a. homogenizing the marigold extract for a time period in the range of 10 minutes to 20 minutes under stirring at a temperature ranging between 50 °C and 60 °C to obtain a homogenized marigold extract;

b. mixing the homogenized marigold extract with hydrocarbon solvent to form a uniform mixture;

c. slowly adding an alcoholic alkali hydroxide solution to the uniform mixture of step (b) with stirring at a temperature ranging between 50 °C and 60 C for a time period in the range of 20 minutes to 45 minutes and continuing stirring after addition of alcoholic alkali hydroxide at a temperature in the range of 60°C to 90 °C for a time period between 60 minutes and 95 minutes to obtain a reaction mixture;

d. analyzing the reaction mixture for completion of de-esterification by HPLC followed by removal of alcohol from the reaction mixture by distillation to obtain a de-esterified substantially alcohol free mixture;

e. diluting the de esterified substantially alcohol free mixture at a temperature in the range of 50 °C to 70 °C with water and cooling at a temperature ranging between 10 °C and 15 °C after dilution to obtain a biphasic mixture comprising an aqueous layer and a hydrocarbon layer;

f. separating the hydrocarbon layer from the aqueous layer of the biphasic mixture of step (e) followed by the centrifugation of the aqueous layer to obtain crystals; and

g. washing the crystals obtained after centrifugation with water followed by vacuum drying to obtain the final product.


2. The process as claimed in claim 1, wherein the plant extract is preferably prepared from mutant marigold flowers.

3. The process as claimed in claim 1 wherein the hydrocarbon solvent is at least one selected from the group of alkanes having carbon atoms greater than 25.

4. The process as claimed in claim 1 wherein the hydrocarbon solvent is liquid paraffin.

5. The process as claimed in claim 1 wherein the alcohol is at least one selected from the group comprising of methanol, ethanol, propanol, propan-2-ol and 1-butanol.

6. The process as claimed in claim 1 wherein the alcohol is preferably ethanol.

7. The process as claimed in claim 1 wherein the alkali hydroxide is at least one selected from the group comprising of lithium hydroxide, sodium hydroxide potassium hydroxide and cesium hydroxide.

8. The process as claimed in claim 1 wherein the alkali hydroxide is preferably potassium hydroxide.

9. The process as claimed in claim 1 wherein the percentage of alkali hydroxide present in the alcohol is in the range of 15% to 30%.

10. The process as claimed in claim 1 wherein the method step (f) optionally involves diluting the separated aqueous layer with water before centrifugation. a temperature ranging between 40 °C and 60 °C for a time period in the range

11. The process as claimed in claim 1 wherein the vacuum drying is carried out at a temperature rangir of 1 hour to 5 hours.

12. The process as claimed in claim 1, wherein the product obtained comprising zeaxanthin crystals has moisture content less than 3%.

13. The process as claimed in claim 1 wherein the percentage of xanthophyll concentration in the product is between 45% and 72%.

14. The process as claimed in claim 1 wherein the product formed has carotenoid profile of 85% to 95% of zeaxanthin.

15. The process as claimed in claim 1 wherein the percentage of zeaxanthin recovery from the input mutant marigold extract is in the range of 55 % to 70%.

16. The product obtained by the process as claimed in claim 1 comprising zeaxanthin.

Documents

Name Date
745-CHE-2011 POWER OF ATTORNEY 11-03-2011.pdf 2011-03-11
745-CHE-2011 FORM-3 11-03-2011.pdf 2011-03-11
745-CHE-2011 FORM-2 11-03-2011.pdf 2011-03-11
745-CHE-2011 FORM-1 11-03-2011.pdf 2011-03-11
745-CHE-2011 CORRESPONDENCE OTHERS 11-03-2011.pdf 2011-03-11
745-CHE-2011 DESCRIPTION (PROVISIONAL) 11-03-2011.pdf 2011-03-11
745-CHE-2011 FORM-5 05-03-2012.pdf 2012-03-05
745-CHE-2011 FORM-2 05-03-2012.pdf 2012-03-05
745-CHE-2011 CORRESPONDENCE OTHERS 05-03-2012.pdf 2012-03-05
745-CHE-2011 ABSTRACT 05-03-2012.pdf 2012-03-05
745-CHE-2011 CLAIMS 05-03-2012.pdf 2012-03-05
745-CHE-2011 FORM-18 30-03-2012.pdf 2012-03-30
745-CHE-2011 CORRESPONDENCE OTHERS 30-03-2012.pdf 2012-03-30
745-CHE-2011 CORRESPONDENCE OTHERS 24-12-2012.pdf 2012-12-24
745-CHE-2011 FORM-1 24-12-2012.pdf 2012-12-24
745-CHE-2011 DESCRIPTION(COMPLETE) 05-03-2012.pdf 2012-03-05
Other Patent Document [05-10-2016(online)].pdf 2016-10-05
745-CHE-2011-FER_SER_REPLY [17-11-2017(online)].pdf 2017-11-17
745-CHE-2011-FER.pdf 2017-06-12
745-CHE-2011-CORRESPONDENCE [17-11-2017(online)].pdf 2017-11-17
745-CHE-2011-CLAIMS [17-11-2017(online)].pdf 2017-11-17
745-CHE-2011-COMPLETE SPECIFICATION [17-11-2017(online)].pdf 2017-11-17
745-CHE-2011-ABSTRACT [17-11-2017(online)].pdf 2017-11-17
745-CHE-2011-HearingNoticeLetter.pdf 2018-05-02
745-CHE-2011-Written submissions and relevant documents (MANDATORY) [14-06-2018(online)].pdf 2018-06-14
745-CHE-2011-Written submissions and relevant documents (MANDATORY) [14-06-2018(online)]-1.pdf 2018-06-14
Correspondence by Agent_Form 26_24-08-2018.pdf 2018-08-24
745-CHE-2011-RELEVANT DOCUMENTS [10-09-2018(online)].pdf 2018-09-10
745-CHE-2011-PETITION UNDER RULE 137 [10-09-2018(online)].pdf 2018-09-10
Marked up Claims_Granted 304432_13-12-2018.pdf 2018-12-13
Abstract_Granted 304432_13-12-2018.pdf 2018-12-13
745-CHE-2011-FORM-26 [28-05-2018(online)].pdf 2018-05-28
Claims_Granted 304432_13-12-2018.pdf 2018-12-13
745-CHE-2011-IntimationOfGrant13-12-2018.pdf 2018-12-13
745-CHE-2011-RELEVANT DOCUMENTS [28-01-2019(online)].pdf 2019-01-28
745-CHE-2011-PatentCertificate13-12-2018.pdf 2018-12-13
745-CHE-2011-RELEVANT DOCUMENTS [20-01-2020(online)].pdf 2020-01-20
745-CHE-2011-RELEVANT DOCUMENTS [08-05-2021(online)].pdf 2021-05-08
Description_Granted 304432_13-12-2018.pdf 2018-12-13
745-CHE-2011 POWER OF ATTORNEY 11-03-2011.pdf 2011-03-11
745-CHE-2011 FORM-2 11-03-2011.pdf 2011-03-11
745-CHE-2011 FORM-1 11-03-2011.pdf 2011-03-11
745-CHE-2011 DESCRIPTION (PROVISIONAL) 11-03-2011.pdf 2011-03-11
745-CHE-2011 FORM-2 05-03-2012.pdf 2012-03-05
745-CHE-2011 ABSTRACT 05-03-2012.pdf 2012-03-05
745-CHE-2011 FORM-5 05-03-2012.pdf 2012-03-05
745-CHE-2011 FORM-18 30-03-2012.pdf 2012-03-30
745-CHE-2011 CORRESPONDENCE OTHERS 30-03-2012.pdf 2012-03-30
745-CHE-2011 DESCRIPTION(COMPLETE) 05-03-2012.pdf 2012-03-05
745-CHE-2011 CORRESPONDENCE OTHERS 24-12-2012.pdf 2012-12-24
745-CHE-2011 FORM-1 24-12-2012.pdf 2012-12-24
745-CHE-2011-FER.pdf 2017-06-12
Other Patent Document [05-10-2016(online)].pdf 2016-10-05
745-CHE-2011-COMPLETE SPECIFICATION [17-11-2017(online)].pdf 2017-11-17
745-CHE-2011-ABSTRACT [17-11-2017(online)].pdf 2017-11-17
745-CHE-2011-HearingNoticeLetter.pdf 2018-05-02
745-CHE-2011-Written submissions and relevant documents (MANDATORY) [14-06-2018(online)]-1.pdf 2018-06-14
745-CHE-2011-PETITION UNDER RULE 137 [10-09-2018(online)].pdf 2018-09-10
Correspondence by Agent_Form 26_24-08-2018.pdf 2018-08-24
Marked up Claims_Granted 304432_13-12-2018.pdf 2018-12-13
Description_Granted 304432_13-12-2018.pdf 2018-12-13
745-CHE-2011 FORM-3 11-03-2011.pdf 2011-03-11
745-CHE-2011-FORM-26 [28-05-2018(online)].pdf 2018-05-28
745-CHE-2011 CORRESPONDENCE OTHERS 11-03-2011.pdf 2011-03-11
745-CHE-2011 CORRESPONDENCE OTHERS 05-03-2012.pdf 2012-03-05
745-CHE-2011 CLAIMS 05-03-2012.pdf 2012-03-05
745-CHE-2011-CLAIMS [17-11-2017(online)].pdf 2017-11-17
745-CHE-2011-FER_SER_REPLY [17-11-2017(online)].pdf 2017-11-17
745-CHE-2011-CORRESPONDENCE [17-11-2017(online)].pdf 2017-11-17
745-CHE-2011-Written submissions and relevant documents (MANDATORY) [14-06-2018(online)].pdf 2018-06-14
745-CHE-2011-IntimationOfGrant13-12-2018.pdf 2018-12-13
745-CHE-2011-RELEVANT DOCUMENTS [28-01-2019(online)].pdf 2019-01-28
Abstract_Granted 304432_13-12-2018.pdf 2018-12-13
745-CHE-2011-PatentCertificate13-12-2018.pdf 2018-12-13
Claims_Granted 304432_13-12-2018.pdf 2018-12-13
745-CHE-2011-RELEVANT DOCUMENTS [27-05-2022(online)].pdf 2022-05-27
745-CHE-2011-RELEVANT DOCUMENTS [20-01-2020(online)].pdf 2020-01-20
745-CHE-2011-RELEVANT DOCUMENTS [08-05-2021(online)].pdf 2021-05-08
745-CHE-2011-RELEVANT DOCUMENTS [10-09-2018(online)].pdf 2018-09-10

Orders

Applicant Section Controller Decision Date URL